It has been well established that the ubiquitin pathway plays an important role as a key regulator of neuronal biology. The ubiquitin pathway enzymes are essential and critical mediators of synaptic adaptability in mature neurons, and dysfunctional ubiquitin pathway enzymes or regulation is associated with diverse neurodegenerative disorders. Deubiquitinating enzymes, or DUBs belong to a family of ~100 primarily single subunit proteins that remove ubiquitin from lysine residues of target proteins. Evidence suggests that that dysregulated or un-regulated DUB(s) can lead to neurodegenerative disease. Most commonly used assays for DUB activity are not suitable for either clinical applications or drug discovery. In this proposal, a novel assay for the DUB family of proteins will be developed. The assay involves a semi-synthetic approach to develop reagents that are extensions of the C-terminus of ubiquitin that is conjugated to target proteins. A library of peptides can be extended from the C-terminus of ubiquitin; the best reagent for a given DUB can be selected from the library since classes of DUBs show different specificity preferences. In contrast to their fate in solution phase assays, entities cleaved by DUBs will be immobilized on a surface, allowing them to be suitable reagents for immobilized DUBs in a multiplex environment. Since the assay can identify a protein as a DUB, it can also uncover DUB interactive proteins or the accumulation of ubquitinated substrates common in neurodegenerative diseases. Thus, the assay will be useful for diagnostic as well as drug discovery purposes. In Phase II, the assay will be expanded to include all known DUBs. The commercial application of this technology is the generation of products for diagnostic and therapeutic uses as applied to DUB-linked medical needs such as neurodegenerative diseases. Proteins modified by another small protein called ubiquitin are key regulators of neuronal biology. Enzymes that catalyse the addition or removal of ubiquitin to target proteins are important regulators of neuronal cell function and are potentially useful for diagnosing various neurological diseases or discovering drugs to treat the diseases. PUBLIC HEALTH RELEVANCE: LifeSensors proposes to develop an assay for one of the major types of ubiquitin pathway enzymes known as DUBs (deubiquitinating enzymes). This assay represents a novel technology that will have potential utility in diagnostics and in the discovery of potent and selective drugs to treat Alzheimer's Disease and other neurological diseases. [unreadable] [unreadable] [unreadable]